1. Field of the Invention
The present invention relates to an immunometric assay using monoclonal antibodies for the detection and/or determination of the levels of multiepitopic antigens, such as, for example, alpha-feto protein or human chorionic gonadotropin.
2. Brief Description of the Background Art
Immunometric assays (also called "sandwich" assays) using monoclonal antibodies for the determination of the presence and/or concentration of antigenic substances in fluids are well known. For example, David et al. U.S. Pat. No. 4,376,110 suggests that this type of assay is useful for the determination of a variety of polyvalent antigenic substances against which antibodies can be produced. David et al. mention, among others, hepatitis A and B, IgE and alpha-fetoprotein. The assays are described in the simultaneous, reverse and forward modes, and specifically exemplified for the determination of IgE.
Alpha-fetoprotein (AFP) is a major serum protein synthesized by fetal liver cells, yolk sac cells, and in trace amounts by the fetal gastrointestinal tract. Reappearance of AFP in adult serum often signals pathologic conditions, particularly the presence of hepatocellular carcinomas (HCC) and germ cell tumors containing yolk sac cell elements. Although existing assays may be successfully used for monitoring treatment of AFP-producing tumors and as an independent prognostic tool, the finding of elevated serum AFP levels in some patients with nonmalignant liver diseases, particularly in acute and chronic viral hepatitis and cirrhosis, has limited the value of such assays as an independent specific test to establish the diagnosis of cancer.
An important goal in current AFP research is to improve the cancer specificity of the test, particularly given the need for early detection of HCC in high risk populations.
Commercial assays for alpha-feto protein have involved the use of polyvalent sera with all of the attendant limitations and problems. Published immunoassays using monoclonal antibodies against AFP include those of Portsman, T. et al., Clin. Chim. Acta. 135:13-22 (1983); Brock, D. J. et al., ibid, 122:353-8 (1982); Uotila, M., et al , J. Imm. Methods, 42:11-15 (1981); Hunter, W. M. et al , ibid, 50:133-144 (1982); Van Heyningen, V., et al, ibid, 50:123-131 (1982); Nomura, M. et al, ibid, 56:13-17 (1983); Nomura, M. et al., ibid, 58:293-300 (1983); Portsmann, B. et al., ibid, 66:173-185 (1984) Engvall et al. in European Patent Application 0048357, published Mar. 31, 1982, also describe a sandwich assay for AFP which may utilize two different monoclonal antibodies, one on a solid phase and the other in soluble labelled form.
These previously described immunoassays for AFP are of two types. First, a two site immunoassay based on two monoclonal antibodies directed against two separate and distinct epitopes, one of these antibodies on a solid phase and the other in soluble labelled form. Second, a three site immunoassay based on three monoclonal antibodies directed against three separate and distinct epitopes, one of these antibodies on a solid phase and both others in soluble labelled form. None of the data has reported a three site immunoassay based on two different monoclonal antibodies directed against two distinct and separate epitopes on said antigen, the first of these antibodies on a solid phase, and the second on the solid phase and in soluble labelled form.
One of the problems with many prior art assays is that heretofore it has not been possible to use serum AFP levels as a screening assay for HCC, since most patients with acute and chronic active hepatitis (with and without cirrhosis) often show AFP elevations up to 1,000 ng/ml, and sometimes greater than 5,000 ng/ml. Thus, the diagnosis of HCC can only be suspected in patients with AFP levels above 400-1,000 ng/ml, and other methods are required to confirm the diagnosis. In addition, often at the time of diagnosis, the size of the tumor is already too large and/or the cirrhosis too far advanced to warrant consideration of surgical resection. In a few patients, the discovery of an elevated AFP has led to curative resections of small tumors.
An acute need therefore still exists in the art for an assay for AFP levels which is selective for patients with HCC and can distinguish over those where the levels of AFP are higher than those related to non-HCC causes, such as cirrhosis or hepatitis.
Human chorionic gonadotropin (HCG) is a hormone secreted by the placenta shortly after the start of pregnancy. It can be measured by radioimmunoassay, and detected in the blood as early as 6 days after conception. Its presence in the urine in early pregnancy is the basis for various laboratory tests for pregnancy, and it can sometimes be detected in the urine as early as 14 days after conception.
Assays for such important antigens as AFP, HCG and others are in constant need of improvement, both in sensitivity and selectivity.